1. Field of the Invention
The present invention generally relates to double stranded RNA phages, and more particularly, to recombinant double stranded RNA phages (hereinafter rdsRP) that express dsRNA-encoded genes in eukaryotic cells and used for the expression of dsRNA expression cassettes encoding passenger genes.
2. Background of the Related Art
Double stranded RNA phage (herein “dsRP”) are atypical compared to other RNA and DNA phage, and more closely resemble members of the reoviridae family [1-5]. The distinguishing attributes of dsRP are a genome comprised of three double-stranded RNA (herein “dsRNA”) segments [2-4,6] and a lipid-containing membrane coat [7-12]. The genomic segments are contained within the nucleocapsid core, which is comprised of the proteins P1, P2, P4, and P7, and is produced by genes encoded on the 7051 bp dsRNA segment, designated “segment L” (GeneBank Accession # AF226851). Synthesis of positive-strand RNA (herein “mRNA”) occurs within the nucleocapsid, which is carried out by RNA-dependent RNA polymerase that may be encoded by gene 2 on segment L, based on sequence similarity to other bacterial RNA polymerases [4,13]. However, gene 7 on segment L also plays a pivotal role in mRNA synthesis [5].
DsRP phi-6, the archetype of this family of dsRNA phage, normally infects Pseudomonas syringae [5], however, more recently isolated dsRP phi-8, phi-11, phi-12 and phi-13 can replicate to some extent in Escherichia coli strain JM109 (American type tissue culture collection (herein “ATCC” # 53323) and O-antigen negative mutants of Salmonella enterica serovar Typhimurium (herein designated “S. typhimurium”) [5,14-16].
By inserting a kanamycin-resistance allele into the M-segment of a dsRP, carrier strains were established and maintained [17]. Through this approach, several of the dsRPs were found to be capable of establishing a carrier state in host cells, in which infectious phage are continuously produced by the carrier strain [17]. The plaque-forming capacity of the phage produced by the carrier strains is maintained for three-five plate passages; however, after additional passages the nascent phage no longer formed plaques on the carrier strain, yet low-levels of infectious phage were still produced [17]. In some instances, a significant number of carrier strains lost the ability to produce infectious phage all together, yet phage dsRNA segments were continuously maintained in the cytosol of such carrier bacteria. The dsRNA from such bacterial strains displayed deletions in one of more of the segments. In one instance a mutant phage lacking the segment-S was isolated from one such carrier strain that had lost the capacity to produce phage [17,18].
The life cycle of the dsRP phi-6 in bacteria has been described [5,11]. Archetype dsRP phi-6 infects host cells by binding to the pilus. The phage then uses the pilus to allow contact with the host cell membrane, thereby resulting in fusion and introduction of the nucleocapsid into the periplasm. The nucleocapsid then is transported into the cytoplasm, an event that requires the endopeptidase activity of protein P5 and the transporting property of protein P8. Interestingly, nucleocapsids that bear a complete P8 shell are capable of spontaneous entry into bacterial protoplasts, resulting in auto-transfection of the bacterial strain from which the protoplasts were prepared [19,20].
Upon entering the cytoplasm, P8 is shed and the remaining nucleocapsid, which contains the three dsRNA segments and possesses RNA-dependent RNA polymerase activity, begins to synthesize mRNA copies of the dsRNA segments L, M and S as shown in FIG. 1. The proteins produced by segment L is mainly associated with procapsid production; segment M is mainly dedicated to the synthesis of the attachment proteins and the segment S produces the procapsid shell protein (P8), the lytic endopeptidase (P5), and the proteins (P9 and P12) involved in the generation of the lipid envelope [12] (FIG. 1).
Packaging of the dsRNA segments occurs in sequential manner, whereby segment S is recognized and taken up by empty procapsids; procapsids containing segment S no longer binds this segment but now are capable of binding and taking up segment M; procapsids that contain segments S and M no longer bind these segments but now are capable of binding and taking up segment L, resulting in the generation of the nucleocapsid. Once the nucleocapsid contains all three single-stranded RNA (herein “ssRNA”) segments synthesis of the negative RNA strands begins to produce the dsRNA segments. The nucleocapsid then associates with proteins 5 and 8 as illustrated in FIG. 1 and finally is encapsulated in the lipid membrane, resulting the completion of phage assembly. Lysis of the host cell is thought to occur through the accumulation of the membrane disrupter protein P10, a product of segment M and requires the endopeptidase P5 [5].
The assembly of RNA polymerase and its activity in dsRP procapsids does not require host proteins, as procapsids purified from an E. coli JM109 derivative that expressed a cDNA copy of segment L are capable of packaging purified ssRNA segments L, M and S [5,19-24]. Following uptake of the ssRNA segments in the above in vitro system, addition of ribonucleotides resulted in negative strand synthesis and the generation of the mature dsRNA segments [5,19-24]. Furthermore, after the completion of dsRNA synthesis P8 associates with nucleocapsids and as indicated above the resultant product is capable of entering bacterial protoplasts and producing a productive infection [19,20].
There are several techniques for introducing nucleic acids into eukaryotic cells cultured in vitro. These include chemical methods (Felgner et al, Proc. Natl. Acad. Sci., USA, 84:7413-7417 (1987); Bothwell et al, Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers Inc., Boston, Mass. (1990), Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y. (1992); and Farhood, Annal. N.Y. Acad. Sci., 716:23-34 (1994)), use of protoplasts (Bothwell, supra) or electrical pulses (Vatteroni et al, Mutn. Res., 291:163-169 (1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62:119-152 (1994); Brothwell et al, supra; and Ausubel, et al, supra), use of attenuated viruses [25-34](Moss, Dev. Biol. Stan., 82:55-63 (1994); and Brothwell et al, supra), as well as physical methods (Fynan et al, supra; Johnston et al, Meth. Cell Biol, 43(Part A):353-365 (1994); Brothwell et al, supra; and Ausubel et al, supra).
Successful delivery of nucleic acids to animal tissue has been achieved by cationic liposomes (Watanabe et al, Mol. Reprod. Dev., 38:268-274 (1994)), direct injection of naked DNA or RNA into animal muscle tissue (Robinson et al, Vacc., 11:957-960 (1993); Hoffman et al, Vacc., 12:1529-1533; (1994); Xiang et al, Virol., 199:132-140 (1994); Webster et al, Vacc., 12:1495-1498 (1994); Davis et al, Vacc., 12:1503-1509 (1994); and Davis et al, Hum. Molec. Gen., 2:1847-1851 (1993); [35,36]), and embryos (Naito et al, Mol. Reprod. Dev., 39:153-161 (1994); and Burdon et al, Mol Reprod. Dev., 33:436-442 (1992)), intramuscular injection of self replicating RNA vaccines [25-28,35,36] or intradermal injection of DNA using “gene gun” technology (Johnston et al, supra).
The ribosomal binding site (RBS) is the site recognized by the ribosome for binding to the 5-prime (herein designated “5′”) end of mRNA molecules. This binding is essential for the translation of mRNA into a protein by the ribosome. In prokaryotes, a defined RBS in the 5′ end of the mRNA molecule that bears a sequence that is complementary to the 3′ end of the small ribosomal RNA molecule (5S rRNA) (Chatteiji et al, Ind. J. Biochem. Biophys., 29:128-134 (1992); and Darnell et al, supra; Lewin, supra; Watson et al, supra; and Watson et al, supra). Thus, in prokaryotes the RBS promotes association of the ribosome with the 5′ end of the nascent mRNA molecule, whereupon translation is initiated at the first initiation codon encountered (i.e. normally the methionine codon AUG) by the mRNA-associated ribosome (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra).
At present, no such recognition pattern has been observed in the 5′ eukaryotic mRNA-ribosome interactions (Eick et al., supra). In addition, prior to initiation of translation of eukaryotic mRNA, the 5′ end of the mRNA molecule is “capped” by addition of methylated guanylate to the first mRNA nucleotide residue (Darnell et al, supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra). It has been proposed that recognition of the translational start site in mRNA by the eukaryotic ribosomes involves recognition of the cap, followed by binding to specific sequences surrounding the initiation codon on the mRNA.
It is possible for cap independent translation initiation to occur and/or to place multiple eukaryotic coding sequences within a eukaryotic expression cassette if a internal ribosome entry site (herein “IRES”) sequence, such as the cap-independent translation enhancer (herein designated “CITE”) derived from encephalomyocarditis virus (Duke et al, J. Virol., 66:1602-1609 (1992)), is included prior to, or between, the coding regions. However, the initiating AUG codon is not necessarily the first AUG codon encountered by the ribosome (Louis et al, Molec. Biol. Rep., 13:103-115 (1988); and Voorma et al, Molec. Biol. Rep., 19:139-145 (1994); Lewin, supra; Watson et al, supra; and Alberts et al, supra). Thus, RBS sequences in eukaryotes are sufficiently divergent from that of prokaryotic RBS such that the two are not interchangeable.
The commercial application of nucleic acid delivery technology to eukaryotic cells is broad and includes delivery of vaccine antigens (Fynan et al, Proc. Natl. Acad. Sci., USA, 90:11478-11482 (1993)), immunotherapeutic agents, and bioactive proteins designed to remedy genetic disorders (Darris et al, Cancer, 74(3 Suppl.): 1021-1025 (1994); Magrath, Ann. Oncol., 5(Suppl 1):67-70 (1994); Milligan et al, Ann. NY Acad. Sci., 716:228-241 (1994); Schreier, Pharma. Acta Helv., 68:145-159 (1994); Cech, Biochem. Soc. Trans., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Long et al, FASEB J., 7:25-30 (1993); and Rosi et al, Pharm. Therap., 50:245-254 1991)).
The delivery of nucleic acids to animal tissue for gene therapy has shown significant promise in experimental animals and volunteers, particularly where a transient effect is required (Nabel, Circulation, 91:541-548 (1995); Coovert et al, Curr. Opin. Neuro., 7:463-470 (1994); Foa, Bill. Clin. Haemat., 2:421-434 (1994); Bowers et al, J. Am. Diet. Assoc., 95:53-59 (1995); Perales et al, Eur. J. Biochem., 226:255-266 (1994); Danko et al, Vacc., 12:1499-1502 (1994); Conry et al, Canc. Res., 54:1164-1168 (1994); and Smith, J. Hemat., 1:155-166 (1992)). Recently, naked DNA vaccines carrying eukaryotic expression cassettes have been used to successfully immunize against influenza both in chickens (Robinson et al, supra) and ferrets (Webster et al, Vacc., 12:1495-1498 (1994)); against Plasmodium yoelii in mice (Hoffman et al, supra); against rabies in mice (Xiang et al, supra); against human carcinoembryonic antigen in mice (Conry et al, supra) and against hepatitis B in mice (Davis et al, supra). These observations open the additional possibility that delivery of nucleic acids to eukaryotic tissue could be used for both prophylactic and therapeutic applications, wherein the prophylactic application has a significant impact in the mortality and/or morbidity of the infectious agent, autoimmune disease or tumor prior to the acquisition of overt clinical disease, and the therapeutic application has a significant impact in the mortality and/or morbidity of the infectious agent, autoimmune disease or tumor following the development of overt clinical disease.
Therefore, there is a need to deliver eukaryotic expression cassettes, encoding endogenous or foreign genes that are vaccines or therapeutic agents to eukaryotic cells or tissue.